Assaying dynamic cell–cell junctional communication using noninvasive and quantitative fluorescence imaging techniques: LAMP and infrared-LAMP

Nature Protocols 4, 94 (2008). doi:10.1038/nprot.2008.219

Authors: Song Yang & Wen-Hong Li

This protocol describes a fluorescence imaging assay, local activation of molecular fluorescent probes (LAMP), for measuring rates of intercellular dye transfer across gap junction channels in intact living cells. The LAMP method consists of four steps: (i) loading cells with a cell-permeable and photo-activatable fluorophore,

A protocol for isolation and culture of mesenchymal stem cells from mouse bone marrow

Nature Protocols 4, 102 (2008). doi:10.1038/nprot.2008.221

Authors: Masoud Soleimani & Samad Nadri

We explain a protocol for straightforward isolation and culture of mesenchymal stem cells (MSCs) from mouse bone marrow (BM) to supply researchers with a method that can be applied in cell biology and tissue engineering with minimal requirements. Our protocol is mainly on the basis

A methodology for the combined in situ analyses of the precursor and mature forms of microRNAs and correlation with their putative targets

Nature Protocols 4, 107 (2008). doi:10.1038/nprot.2008.215

Authors: Gerard J Nuovo, Terry S Elton, Patrick Nana-Sinkam, Stefano Volinia, Carlo M Croce & Thomas D Schmittgen

There are relatively few protocols described for the in situ detection of microRNA (miRNA) and they often use cryostat sections, signal amplification and hybridization or washes of 50–60 °C. This protocol describes in situ miRNA detection that can be done in paraffin-embedded, formalin-fixed

Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources

Nature Protocols 4, 44 (2008). doi:10.1038/nprot.2008.211

Authors: Da Wei Huang, Brad T Sherman & Richard A Lempicki

DAVID bioinformatics resources consists of an integrated biological knowledgebase and analytic tools aimed at systematically extracting biological meaning from large gene/protein lists. This protocol explains how to use DAVID, a high-throughput and integrated data-mining environment, to analyze gene lists derived from high-throughput genomic experiments. The

Engineering complex-type N-glycosylation in Pichia pastoris using GlycoSwitch technology

Nature Protocols 4, 58 (2008). doi:10.1038/nprot.2008.213

Authors: Pieter P Jacobs, Steven Geysens, Wouter Vervecken, Roland Contreras & Nico Callewaert

Here we provide a protocol for engineering the N-glycosylation pathway of the yeast Pichia pastoris. The general strategy consists of the disruption of an endogenous glycosyltransferase gene (OCH1) and the stepwise introduction of heterologous glycosylation enzymes. Each engineering step results in the

Functional transient genetic transformation of Arabidopsis leaves by biolistic bombardment

Nature Protocols 4, 71 (2008). doi:10.1038/nprot.2008.217

Authors: Shoko Ueki, Benoît Lacroix, Alexander Krichevsky, Sondra G Lazarowitz & Vitaly Citovsky

Transient gene expression is an indispensable tool for studying functions of gene products. In the case of plants, transient introduction of genes by Agrobacterium infiltration is a method of choice for many species. However, this technique does not work efficiently in Arabidopsis leaf

Primary support cultures of hippocampal and substantia nigra neurons

Nature Protocols 4, 78 (2008). doi:10.1038/nprot.2008.199

Authors: Thomas Fath, Yazi D Ke, Peter Gunning, Jürgen Götz & Lars M Ittner

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. Their use is limited, as culturing at low density is often not possible or is dependent on sophisticated methods. Here we present a novel method for culturing embryonic

Orthotopic mouse lung transplantation as experimental methodology to study transplant and tumor biology

Nature Protocols 4, 86 (2008). doi:10.1038/nprot.2008.218

Authors: Alexander S Krupnick, Xue Lin, Wenjun Li, Mikio Okazaki, Jiaming Lai, Seiichiro Sugimoto, Steven B Richardson, Christopher G Kornfeld, Joel R Garbow, G Alexander Patterson, Andrew E Gelman & Daniel Kreisel

Unlike transplantation of other solid organs, vascularized mouse lung transplantation has only recently been developed. In this protocol, we describe a detailed method for performing a vascularized and aerated mouse orthotopic lung transplant, which to date represents the most physiological mouse model of lung transplantation.

Protein structure homology modeling using SWISS-MODEL workspace

Nature Protocols 4, 1 (2008). doi:10.1038/nprot.2008.197

Authors: Lorenza Bordoli, Florian Kiefer, Konstantin Arnold, Pascal Benkert, James Battey & Torsten Schwede

Homology modeling aims to build three-dimensional protein structure models using experimentally determined structures of related family members as templates. SWISS-MODEL workspace is an integrated Web-based modeling expert system. For a given target protein, a library of experimental protein structures is searched to identify suitable templates.

Separation of sedimentary micron-sized particles for palaeoceanography and calcareous nannoplankton biogeochemistry

Nature Protocols 4, 14 (2008). doi:10.1038/nprot.2008.200

Authors: Fabrice Minoletti, Michaël Hermoso & Vincent Gressier

A protocol is described for separating sub-20μm-sized particles contained in sedimentary rocks into size fractions. Geochemical data from manually isolated foraminifera are commonly used in the interpretation of marine palaeoenvironments; problems associated with the isolation of calcareous nannofossils hampers their geochemical exploitation. However, geochemistry performed

Dictyostelium discoideum: a model host to measure bacterial virulence

Nature Protocols 4, 25 (2008). doi:10.1038/nprot.2008.212

Authors: Romain Froquet, Emmanuelle Lelong, Anna Marchetti & Pierre Cosson

Dictyostelium amoebae have been used as a host model to measure virulence of a wide range of bacterial pathogens. The simple protocol described here takes advantage of the ability of Dictyostelium to grow and form plaques on a lawn of nonpathogenic bacteria but

Immunodesign of experimental sepsis by cecal ligation and puncture

Nature Protocols 4, 31 (2008). doi:10.1038/nprot.2008.214

Authors: Daniel Rittirsch, Markus S Huber-Lang, Michael A Flierl & Peter A Ward

Sepsis remains a prevalent clinical challenge and the underlying pathophysiology is still poorly understood. To investigate the complex molecular mechanisms of sepsis, various animal models have been developed, the most frequently used being the cecal ligation and puncture (CLP) model in rodents. In this model,

Northern blot analysis for detection and quantification of RNA in pancreatic cancer cells and tissues

Nature Protocols 4, 37 (2008). doi:10.1038/nprot.2008.216

Authors: Sylvia Streit, Christoph W Michalski, Mert Erkan, Jörg Kleeff & Helmut Friess

Investigation of gene expression significantly contributes to our knowledge of the regulation and function of genes in many areas of biology. In this protocol, we describe how northern blot analysis is used to identify gene expression patterns at the RNA level in human cancer cells

Expression cloning and radiotracer uptakes in Xenopus laevis oocytes

Nature Protocols 3, 1975 (2008). doi:10.1038/nprot.2008.151

Author: Daniel Markovich

This protocol describes the method of expression cloning of heterologous proteins using Xenopus laevis oocytes and the functional characterization of membrane proteins using radiotracer assays. It can be used to isolate proteins for which sequence data is unavailable and to characterize the functions of

Cryopreservation and banking of mammalian cell lines

Nature Protocols 3, 1981 (2008). doi:10.1038/nprot.2008.190

Authors: Glyn N Stacey & John R Masters

This protocol describes the principles and methods used for the preparation of cryopreserved cell stocks. Following these procedures will ensure the availability of reproducible cultures for use within a single laboratory at different times and for different collaborating laboratories. Although the basic principle is simple,

Dual fluorescence labeling of surface-exposed and internal proteins in erythrocytes infected with the malaria parasite Plasmodium falciparum

Nature Protocols 3, 1990 (2008). doi:10.1038/nprot.2008.196

Authors: Dominique C Bengtsson, Kordai M P Sowa & David E Arnot

There is a need for improved methods for in situ localization of surface proteins on Plasmodium falciparum–infected erythrocytes to help understand how these antigens are trafficked to, and positioned within, the host cell membrane. This protocol for confocal immunofluorescence microscopy combines surface antigen

A protocol for studying the kinetics of RNA within cultured cells: application to ribosomal RNA

Nature Protocols 3, 1997 (2008). doi:10.1038/nprot.2008.198

Authors: Marc Thiry, Françoise Lamaye, Nicolas Thelen, Aurore Chatron-Colliet, Nathalie Lalun, Hélène Bobichon & Dominique Ploton

This protocol describes a nonisotopic method for high-resolution investigation of the kinetics of RNA within the cell. This involves the incorporation of bromouridine-5′-triphosphate into RNA of living cells by lipofection followed by immunocytological detection of BrRNAs. The use of the same antibody identified either with

A sensitive recombinant cell-based bioluminescent assay for detection of androgen-like compounds

Nature Protocols 3, 1895 (2008). doi:10.1038/nprot.2008.189

Authors: Elisa Michelini, Luca Cevenini, Laura Mezzanotte, Piia Leskinen, Marko Virta, Matti Karp & Aldo Roda

We report a step-by-step protocol describing how to develop and use a yeast-based bioassay for androgen-like compounds. Saccharomyces cerevisiae cells are genetically engineered to express the human androgen receptor (hAR) and the bioluminescent (BL) reporter gene luciferase (from Photinus pyralis) under the control

Methylation-sensitive high-resolution melting

Nature Protocols 3, 1903 (2008). doi:10.1038/nprot.2008.191

Authors: Tomasz K Wojadcz, Alexander Dobrovic & Lise Lotte Hansen

The base composition of PCR products derived from sodium bisulfite-modified templates is methylation dependent. Hence, methylated and unmethylated, PCR products show different melting profiles when subjected to thermal denaturation. The methylation-sensitive high-resolution melting (MS-HRM) protocol is based on the comparison of the melting profiles of

In vitro growth and analysis of Candida biofilms

Nature Protocols 3, 1909 (2008). doi:10.1038/nprot.2008.192

Authors: Jyotsna Chandra, Pranab K Mukherjee & Mahmoud A Ghannoum

Evaluation of fungal biofilm formation can be performed using several techniques. In this protocol, we describe methods used to form Candida biofilms on three different medical device substrates (denture strips, catheter disks and contact lenses) to quantify them and to evaluate their architecture and

A C. elegans-based, whole animal, in vivo screen for the identification of antifungal compounds

Nature Protocols 3, 1925 (2008). doi:10.1038/nprot.2008.193

Authors: Emmanouil Tampakakis, Ikechukwu Okoli & Eleftherios Mylonakis

Traditional antimicrobial screens focus on compounds that block the growth of microbial organisms. A new Caenorhabditis elegans-based bioassay can be used for the identification of antifungal compounds that are effective against Candida albicans. According to the protocol, adult nematodes are infected with C.

In vivo evaluation of human hematopoiesis through xenotransplantation of purified hematopoietic stem cells from umbilical cord blood

Nature Protocols 3, 1932 (2008). doi:10.1038/nprot.2008.194

Authors: Christopher Y Park, Ravindra Majeti & Irving L Weissman

Establishment of robust xenograft models is critical to studying human hematopoiesis in a physiologic setting. Using a recently developed immunodeficient mouse strain, we have established long-term multilineage human grafts and demonstrated their serially transplantability using limited numbers of purified human hematopoietic stem cells (HSCs). Herein,

Methods for generating and colonizing gnotobiotic zebrafish

Nature Protocols 3, 1862 (2008). doi:10.1038/nprot.2008.186

Authors: Linh N Pham, Michelle Kanther, Ivana Semova & John F Rawls

Vertebrates are colonized at birth by complex and dynamic communities of microorganisms that can contribute significantly to host health and disease. The ability to raise animals in the absence of microorganisms has been a powerful tool for elucidating the relationships between animal hosts and their

A protocol for unraveling gene regulatory networks

Nature Protocols 3, 1876 (2008). doi:10.1038/nprot.2008.187

Authors: Stefan C Materna & Paola Oliveri

Regulatory genes form large networks that are fundamental to the developmental program. The protocol presented here describes a general approach to assemble maps of gene regulatory networks (GRNs). It combines high-resolution spatio-temporal profiling of regulatory genes, strategies to perturb gene expression and quantification of perturbation

Efficient derivation of functional dopaminergic neurons from human embryonic stem cells on a large scale

Nature Protocols 3, 1888 (2008). doi:10.1038/nprot.2008.188

Authors: Myung-Soo Cho, Dong-Youn Hwang & Dong-Wook Kim

Cell-replacement therapy using human embryonic stem cells (hESCs) holds great promise in treating Parkinson's disease. We have recently reported a highly efficient method to generate functional dopaminergic (DA) neurons from hESCs. Our method includes a unique step, the formation of spherical neural masses (SNMs), and